Obesity is defined as a condition of abnormal or excessive fat accumulation that may impair health and results from an energy imbalance in which energy intake exceeds energy expenditure. Obesity was not a serious health problem in the past, but with economic growth, the obese population is increasing with increasing economic affluence, and the number of various diseases related to obesity is also increasing. According to the report of the World Health Organization (WHO), over 1.5 billion adults worldwide are overweight, over 500 million of them are obese, and the obesity population increased by about twice between 1980 and 2008 (World Health Organization, Fact sheet on obesity and overweight, 2011). Not only in high-income countries, but also in low-income countries, the percentage of obese people is currently increasing. Overweight and obesity are responsible for increasing blood pressure and cholesterol levels and cause or worsen various diseases. In addition, the obesity problem is more serious in children or teenagers, increases the incidence of diabetes, heart diseases, hypertension or hyperlipidemia, and can also cause deaths or disabilities.
As described above, obesity is a global disease and social problem, but in the past, it was believed that obesity could be overcome by individual efforts, and thus no particular emphasis was placed on the treatment of obesity. However, obesity is not easy to treat, because it is a complex disease associated with the mechanisms of appetite control and energy metabolism. Accordingly, the treatment of obesity requires not only the patient's own efforts, but also a method capable of treating abnormal mechanisms associated with appetite control and energy metabolism. Thus, efforts have been made to develop drugs for treating obesity.
As a result of such efforts, drugs, including Rimonabant (Sanofi-Aventis), Sibutramine (Abbott), Contrave (Takeda), Orlistat (Roche) and the like, were developed, but these drugs have shortcomings in that they show fetal side effects or have an insufficient effect on the treatment of obesity. It was reported that Rimonabant (Sanofi-Aventis) showed disorders of the central nervous system, Sibutramine (Abbott) and Contrave (Takeda) showed cardiovascular side effects, and Orlistat (Roche) showed a weight reduction effect of only about 4 kg when administered for 1 year. Thus, there are currently little or no obesity therapeutic agents that can be safely prescribed for obesity patients.
Recently, glucagon derivatives have received much attention. Glucagon is produced by the pancreas when blood glucose levels start to drop due to medications, diseases, hormone or enzyme deficiencies, or the like. Glucagon functions to stimulate liver cells to break down stored glycogen into glucose which is then released into the blood to raise the blood glucose level to a normal level. In addition to the effect of increasing the blood glucose level, glucagon was reported to suppress appetite and activate hormone-sensitive lipase (HSL) of adipocytes to facilitate lipolysis, thereby showing anti-obesity effects. Among the glucagon derivatives, glucagon-like peptide-1 (GLP-1) is under development as a therapeutic agent for reducing hyperglycemia in diabetic patients and functions to stimulate insulin synthesis and secretion, inhibit glucagon secretion, suppress gastric emptying, increase glucose utilization and inhibit food intake. It is known that exendin-4 that is isolated from lizard venom has an amino acid homology of about 50% with GLP-1 and activates the GLP-1 receptor to reduce hyperglycemia in diabetic patients. However, obesity therapeutic drugs, including GLP-1, were reported to cause side effects such as vomiting and nausea.
Thus, as an alternative to GLP-1, oxyntomodulin capable of binding to both receptors for two peptides (GLP-1 and glucagon) is receiving attention. Oxyntomodulin is a peptide made from pre-glucagon, a precursor of glucagon, and is a potent anti-obesity agent, because it inhibits food intake, like GLP-1, promotes satiety, and shows lipolytic activity, like glucagon.
Based on the dual function of the oxyntomodulin peptide, studies on the development of drugs for the treatment of obesity have been actively conducted. For example, Korean Patent Registration No. 925017 discloses an oral, parenteral, mucosal, rectal, subcutaneous or transdermal pharmaceutical composition for treating human obesity, which comprises oxyntomodulin as an active ingredient. However, it was reported that obesity therapeutic agents comprising oxyntomodulin have a short in vivo half-life and show a low effect on the treatment of obesity, even when these are administered at a high dose three times a day. Thus, efforts have been made to increase the in vivo half-life or obesity-treating effect of oxyntomodulin by modifying oxyntomodulin.
For example, the dual agonist oxyntomodulin (Merck) is obtained by substituting L-serine with D-serine at amino acid 2 of oxyntomodulin to increase resistance to dipeptidyl peptidase-IV (DPP-IV) and by attaching a cholesterol moiety to the C terminal to increase the blood half life. ZP2929 (Zealand) is obtained by substituting L-serine with D-serine at amino acid 2 of oxyntomodulin to increase resistance to DPP-IV, substituting arginine with alanine at amino acid 17 to increase resistance to protease, substituting methionine with lysine at amino acid 27 to increase oxidative stability, and substituting glutamines at amino acids 20 and 24 and asparagine at amino acid 28 with aspartic acid, alanine and serine, respectively, to increase deamidation stability. The dual agonist oxyntomodulin (Merck) has an increased in vivo half-life of 1.7 hours, which is longer than the half-life (8-12 minutes) of native oxyntomodulin, but it still has a very short in vivo half-life and is administered at a very high dose of several mg/kg. Thus, oxyntomodulin or derivatives thereof have two big disadvantages, that is, a short half life and low medicinal effects. Due to these disadvantages, they should be administered daily at high doses. In order to overcome these disadvantages, a method was studied to increase the blood half-life of oxyntomodulin while maintaining the in vivo activity thereof, and as a result, an oxyntomodulin derivative was developed. In addition, using this technology, a non-peptidyl polymer was prepared by conjugating a carrier to the oxyntomodulin derivative, and it was found that the protein conjugate can show a better anti-obesity effect as a result of increasing the blood half-life thereof while maintaining the in vivo activity (Korean Patent Application No. 10-2012-0064110).
Generally, proteins and peptides have a very short half-life, and undergo denaturation such as precipitation by aggregation of monomers, and adsorption on the surfaces of vessels, upon exposure to various factors such as unfavorable temperatures, water-air interface, high pressure, physical/mechanical stress, organic solvents and microbial contamination. This denaturation is irreversible, and thus the denatured proteins and peptides lose intrinsic physicochemical properties and physiologically active effects. In addition, proteins and peptides are unstable and susceptible to extrinsic factors such as temperature, humidity, oxygen, UV rays or the like to undergo physical or chemical changes including association, polymerization or oxidation, resulting in substantial loss of activity (Korean Patent Registration No. 10-0389726).
Furthermore, the adsorbed proteins and peptides are easily aggregated by the denaturation process, and the denatured proteins and peptides, when administered to the human body, act as the cause of antibody formation in the human body, and for this reason, the proteins and peptides should be administered in a sufficiently stable form. Accordingly, various methods for preventing the denaturation of proteins and peptides in solution have been studied (John Geigert, J. Parenteral Sci. Tech., 43, No 5, 220-224, 1989; David Wong, Pharm. Tech. Oct. 34-48, 1997; Wei Wang., Int. J. Pharm., 185, 129-188, 1999; Willem Norde, Adv. Colloid Interface Sci., 25, 267-340, 1986; Michelle et al., Int. J. Pharm. 120, 179-188, 1995).
Lyophilization is applied to some protein and peptide drugs to achieve the goal of stability. However, lyophilized products are inconvenient in that they must be re-dissolved in injection water for use. In addition, in the case of lyophilization, massive investment on large-capacity freeze-driers or the like is required, because the lyophilization process is included in the production processes. Further, a method for producing powdered proteins and peptides using a spray drier is also being used, but in this case, economic efficiency is decreased due to a low yield, and exposure to high temperatures can adversely affect the stability of the proteins.
In order to overcome such limitations, studies have been conducted in which stabilizers were added to proteins and peptides in solution to suppress the physicochemical changes of the proteins and peptides while maintaining the in vivo efficiency thereof even upon long-term storage. Human serum albumin, a kind of protein, has been widely used as a stabilizer for various protein drugs, and the performance thereof has been proven (Edward Tarelli et al., Biologicals (1998) 26, 331-346).
A process for purifying human serum albumin includes inactivating biological contaminants such as mycoplasma, prion, bacteria and virus and screening or examining one or more biological contaminants or pathogens. However, there is always the risk that patients will be exposed to the biological contaminants that are not completely removed or inactivated. For example, the screening process includes examining whether human blood from donators contains a certain virus, but this process is not always reliable. Particularly, a specific virus existing in a very small number of donators cannot be detected.
Different proteins may be gradually inactivated at different rates under different conditions during storage, due to their chemical differences. That is to say, the extension of the storage term by a stabilizer is not identical for different proteins. For this reason, the suitable ratio, concentration and kind of stabilizer that is used to provide storage stability vary depending on the physicochemical properties of the target protein. When stabilizers are used in combination, they may cause adverse effects different from desired effects due to the competition and interaction therebetween. Further, because the nature or concentration of proteins may change during storage, the stabilizers used may show effects different from those intended. Thus, a great amount of effort and precautions are required to stabilize proteins in solution.
Particularly, a conjugate of oxyntomodulin and immunoglobulin Fc is a conjugate in which oxyntomodulin that is a physiologically active peptide is linked to an immunoglobulin Fc region. Thus, because the molecular weight and volume of the conjugate certainly differ from those of native oxyntomodulin, a special composition for stabilizing the protein is required.
Further, because oxyntomodulin (that is a physiologically active peptide) and the immunoglobulin Fc region are peptides or proteins having different physicochemical properties, they should be simultaneously stabilized. However, as described above, different proteins or proteins may be gradually inactivated at different rates under different conditions during storage, due to their chemical differences, and when stabilizers suitable for proteins or peptides are used in combination, they may cause adverse effects different from the desired effects due to the competition and interaction therebetween. Thus, in the case of a long-lasting oxyntomodulin conjugate, there is much difficulty in finding a composition for simultaneously stabilizing oxyntomodulin, which is a physiologically active peptide, and the immunoglobulin Fc region.
Under such circumstances, the present inventors have made extensive efforts to provide a stable liquid formulation that can be stored for a long period of time without concern about viral contamination, and as a result, have found that a stabilizer, which includes a buffer, a sugar alcohol and a nonionic surfactant and may further include an additive, such as an isotonic agent or an amino acid, and a preservative for repeated use, can increase the stability of a long-lasting oxyntomodulin derivative, and a cost-effective and stable liquid formulation can be prepared using the stabilizer, thereby completing the present invention.